Information concerning protein-protein interactions and protein-compound interactions is very useful in discovering novel drug targets and/or novel candidate compounds for pharmaceutical preparations. Through a comprehensive analysis of protein functions, protein functions can be analyzed with the use of proteins expressed on a minute scale. This is important in terms of cost and throughput. In order to attain the aforementioned information or to implement protein function analysis with the use of proteins expressed on a minute scale, apparatuses that analyze interactions in real time based on the principle of surface plasmon resonance (SPR) without the use of a radioisotope, such as the Biacore 3000 (Biacore), have been used in recent years.
In such SPR-based interaction analysis, either the proteins or compounds to be analyzed are immobilized on a sensor chip, other proteins or compounds are allowed to react on the sensor chip, and changes in masses resulting from protein-protein or protein-compound interactions are detected as an SPR signal. Since this SPR-based interaction analysis is a technique for highly sensitive function analysis, the amount of proteins required therefor is advantageously small.
In SPR-based interaction analysis, for example, a method wherein amino groups of proteins are allowed to couple to carboxyl groups on a sensor chip under conditions of low pH and low salt concentration (i.e., amine coupling) is used when immobilizing proteins on the sensor chip. Under such conditions, however, proteins are likely to be deactivated, and acidic proteins cannot be immobilized. When a tag such as a histidine tag is used, a wide variety of histidine tag fusion proteins can be immobilized on the sensor chip. Such immobilization is unstable, and interaction analysis is impossible. In order to overcome such drawbacks, therefore, the present inventors continuously conducted tag-mediated binding and amine coupling of tag fusion proteins, developed a technique whereby almost all types of proteins could be stably immobilized on a sensor chip at physiological pH and physiological salt levels (affinity amine coupling), and already filed for a patent with regard to the same (JP Patent Application No. 2002-335334).
Protein expression systems that can express proteins regardless of species have been developed (e.g., a wheat germ cell-free system). In such expression systems, however, the expression levels of the target proteins are often very low. When small amounts of target proteins are immobilized on a sensor chip via the aforementioned affinity amine coupling technique, contaminating proteins are simultaneously coupled. This often yields unsatisfactory results in terms of an S/N ratio in the interaction assay.